T. H. Pennington

Molecular characterization of rifampin-resistant Neisseria meningitidis.

Primers were designed to amplify the rpoB gene of Neisseria meningitidis. The region of the gene amplified covered clusters I and II of the rifampin resistance (Rifr) mutation sites identified in Escherichia coli. DNAs from six Rifr isolates and 21 rifampin-susceptible isolates from the United Kingdom representing a number of serogroups were amplified and sequenced. All six Rifr isolates had identical DNA sequences and the same amino acid change, a His to an Asn change at position 35 (H35N). This His residue is equivalent to the His residue at position 526 in E. coli, one of the known Rifr mutation sites. DNAs from an additional six Rifr mutations generated in vitro were amplified and sequenced. Three had H35Y changes, one had an H35R change, one had an H35N change and one had an S40F change. The predominance of mutations at the His residue at position 35 in Rifr N. meningitidis isolates suggests that it plays a critical role in the selection of antibiotic-resistant variants. All six Rifr isolates belonged to the same clonal group when analyzed by restriction enzyme analysis and pulsed-field gel electrophoresis. These data suggest that a single clone of Rifr N. meningitidis is present and widespread throughout the United Kingdom. Images

Molecular typing methods for Neisseria meningitidis

Neisseria meningitidis is an important pathogen because it causes life-threatening infections. The rapid course of meningococcal disease and the capacity of some serogroups to cause large-scale epidemics necessitates the use of sensitive, reliable and rapid typing methods to characterise strains. Molecular typing techniques for N. meningitidis are used for epidemiological purposes to investigate outbreaks and the spread of organisms and to examine the population genetic structure of the organism to understand better its variation and evolution. Many investigators have employed molecular typing methods and shown that meningococcal disease is associated with a variety of different epidemiological patterns. The choice of a typing method is dependent upon the epidemiological questions to be answered and on the population genetics of the organism under investigation. With highly clonal populations comprising independent non-recombining lineages such as serogroup A meningococci, ribotyping, multilocus enzyme electrophoresis (MLEE), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR with arbitrary primers (RAPD) or with other gene-based primers each provides a constant measure of the relationship between strains. A more restricted portfolio of molecular methods - PFGE, MLEE and MLST - is appropriate for the investigation of less clonal serogroup B and C meningococci from localised outbreaks. If a thorough evaluation of the overall population is sought to determine the relationship between new isolates and members of hyper-endemic clonal complexes then quantitative methods such as MLEE and MLST are necessary. Several PCR-based methods are used for the detection and typing of meningococcal strains, many requiring rigorous standardisation before they can be considered suitable for rapid and reliable differentiation between clones. This review examines strain characterisation by molecular techniques and non-culture-based subtyping of meningococci in clinical specimens. It assesses the importance of these techniques and examines the epidemiological questions that they answer and also their limitations.

Staphylococcal whole-cell polypeptide analysis: evaluation as a taxonomic and typing tool.

Whole-cell-polypeptide profiles obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were used in conjunction with the API-Staph technique to identify different strains of Staphylococcus aureus, S. epidermidis, S. saprophyticus and S. capitis. Complete concordance of results from both techniques was achieved with all strains examined. Visual analysis of the polypeptide patterns and comparison by use of the coefficient of Dice showed minor differences in band pattern between strains of the same species but each species produced a pattern distinguishable from that of any other. The results suggest that although SDS-PAGE can be used to identify staphylococcal species, this type of analysis will not readily provide the basis for a typing method.

Allelic variation in the Helicobacter pylori flagellin genes flaA and flaB : its consequences for strain typing schemes and population structure

Extensive DNA sequence diversity was noted in Helicobacter pylori flagellin genes flaA and flaB. PCR amplified sequences from 49 isolates were digested with AluI, HindIII, MboI or MspI, the resultant patterns were compared between the different isolates and these used to differentiate the isolates from each other. Evidence that the extensive diversity that was found in these genes is the result of reassortment of sequences between strains in the bacterial population is presented, such that a comparatively small number of individual sequence mutations can recombine together in random combinations to form a greater number of distinct alleles. Geographical differences in the predominant patterns in the flaA alleles were also observed and could reflect regional differences either in the human host population or in the bacterial population. In view of the genetic complexity of this species, molecular typing schemes designed to identify related strains may falsely associate strains if the methods do not characterize sufficient genetic sites to exclude chance associations of genetic markers in strains which are actually not closely related to each other.

The use of a nested polymerase chain reaction for detecting Pneumocystis carinii from lung and blood in rat and human infection

A nested polymerase chain reaction (PCR) assay was developed to detect both rat- and human-derived Pneumocystis carinii DNA. The nested PCR product was 125 bp long and was representative of part of the gene coding for the large subunit of mitochondrial ribosomal RNA. Twenty serial blood samples and 24 tissues from six immunosuppressed Sprague-Dawley rats were examined by nested PCR. All lung samples were positive by PCR and Toluidine blue O staining. Buffy coat samples and all the other tissues were PCR-negative during up to 6 weeks of immunosuppression. Thirty-five clinical bronchoalveolar lavage, induced sputum or tracheal aspirate samples from human patients were tested. Twelve of 35 were positive by both PCR and indirect fluorescence assay (IFA) and 19 of 35 were both PCR- and IFA-negative. Four of 35 were IFA-negative but PCR-positive and there were good responses in these patients to specific therapy, indicating that PCR may be more useful than IFA in clinical samples. P. carinii DNA was not detected in three blood samples. The nested PCR is a sensitive and specific DNA amplification method suitable for the routine diagnosis of P. carinii in human respiratory samples.

Variation in length and sequence of porin (ompP2) alleles of non‐capsulate Haemophilus influenzae

Length variations of Haemophilus influenzae outer membrane porin protein P2 were found at the DNA and protein levels, notably in non‐capsulate strains. Protein length, measured by SDS‐polyacrylamide gel electrophoresis, was found to correlate with the length of the gene, measured by polymerase chain reaction amplification, and ranged from 35–42 kDa and 970–1090 nucleotides, respectively. This represents a length variation of some 15%. The genetic location of these variations was studied by restriction enzyme mapping 10 of the non‐capsulate strains revealing further polymorphisms at the DNA level. All 10 strains were distinct and differed from a type b strain. The conservation and assortment of the different restriction sites in the alleles is discussed in relation to the very great diversity previously described for this protein and of the whole genome itself in non‐capsulate strains. The roles of selection, horizontal gene transfer, and transformation in generating this diversity are discussed.

Typing of strains of Staphylococcus aureus by Western Blot analysis of culture supernates.

Extracellular proteins produced by Staphylococcus aureus strains were examined by Western Blot analysis with blood donor plasma as a source of antibodies. Comparison of epidemiologically related strains showed strong concordance between plot pattern and phage type.

Incidence and spread of Haemophilus influenzae on an Antarctic base determined using the polymerase chain reaction

A PCR-based method of detecting Haemophilus influenzae in cultures inoculated from throat swabs was evaluated using samples from groups of laboratory staff and medical students and then applied to samples originating from the closed human community of an Antarctic research station. Suitable PCR primers to an H. influenzae gene (ompP2) were used to amplify the gene from DNA preparations made from mixed growth on chocolate agar with added vancomycin. PCR product was reamplified and subjected to restriction endonuclease digestion to allow temporal and spatial mapping of strains over an 8-month period. Eleven different strains of H. influenzae were detected. One particular strain was detected in a third of the base members.

Western blot analysis of staphylococcal antibodies present in human sera during health and disease

IgG antibodies directed against Staphylococcus aureus were examined by Western blotting in sera from 15 healthy individuals isolated for a year in Antarctica. Sera reacted with many staphylococcal antigens in whole-cell extracts and individuals showed unique and unchanging blot profiles. The IgG and IgM profiles of patients with deep-seated staphylococcal infections were also examined by Western blotting. Anti-staphylococcal IgM antibodies that reacted with an antigen of apparent molecular mass 31 X 10(3) were present in all patients with staphylococcal disease, and were absent from, or detected in much smaller amounts in, control sera.

Rapid methods in bacterial DNA fingerprinting.

Summary: The characterization and comparison of isolates of bacterial species by comparing restriction enzyme digests of their chromosomal DNA (fingerprints) is highly discriminatory for different strains and allows similarities between them to be readily determined. However, the utility of the technique is dependent on the selection of appropriate restriction enzyme(s) and on the method of determining the similarities between the fingerprints generated. We report here a system which circumvents these two problems. The restriction enzyme is selected from amongst those which have a suitable frequency of restriction for given enzyme-genome combinations. The frequencies of restriction enzyme recognition sites are calculated from the frequencies of di- and trinucleotides in sequenced genes from the species of interest using Markov chain analysis. Fingerprints are compared by dividing them up into sections with DNA size standards, scoring the number of bands in a few of these sections, and comparing these scores (numerical profiles) to establish similarities. In this way a single electrophoretic gel yields easily analysable data which can be compared with data from other gels. The time from the acquisition of bacterial isolates to their final characterization is much reduced in comparison to existing methods.